The Golgi complex consists of a series of compartments that constitute the hub for intracellular membrane traffic. It functions as the cell's main sorting station where biosynthetic products and recycling membrane traffic are sorted for delivery to multiple destinations. Little is known at present about how its compartmentalization is maintained, how this traffic is controlled, and what signals and signal transduction processes are involved. Recently it has become clear that heterotrimeric G proteins, as well as, small GTPases (Rab, ARF, Sar) are involved in these processes. The overall goal of Project 1 is to understand the role of heterotrimeric proteins in maintaining Golgi organization and in controlling vesicular traffic through the Golgi. During the current grant period we have identified a new protein called GAIP involve din the negative regulation of G protein signaling. Characterization of GAIP has revealed that it is membrane anchored GAP for Gai subunits. It is a member of the newly described RGS (Regulators of G Protein Signaling) family which now has approximately 20 members. The Specific Aims that we now wish to pursue are to: 1) Determine the localization and properties of GAIP. This will be accomplished by immunocytochemistry, cell fractionation and immunoisolation of the cell compartment in which GAIP is located. We will also assess GAIP's effects on trafficking along the exocytic pathway and investigate the role of palmitoylation and phosphorylation on its membrane association, localization and function. 2) Analyze the localization and functions of other, selected RGS family members in relationship to GAIP with an eye to determining with which particular cell membrane they are associated. The goal here is to establish whether expression of RGS family membranes is cell type-specific, tissue-specific, or organelle-specific. 3) Characterize CALNUC (Nucleobindin)--another protein that interacts with Gai3. We have localized CALNUC to the Golgi apparatus cytosol and now plan investigate the relationship between these two pools. These studies should shed light on the role of RGS proteins and CALNUC in transport along the exocytic pathway and the mechanisms and pathways of G protein signaling from intracellular compartments.